Plant dihydroorotase

ABSTRACT

The present invention relates to a DNA encoding a polypeptide with dihydroorotase (EC 3.5.2.3) activity. Also, the invention relates to the use of this nucleic acid for the generation of an assay system.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Divisional application of application Ser. No. 10/070,277filed on Mar. 6, 2002 now U.S. Pat. No. 7,320,877, the entire disclosureof which is hereby incorporated by reference, which is a national stageentry of PCT/EP00/08581 filed on Sep. 2, 2000.

The present invention relates to the identification of plantdihydroorotase as a novel target for herbicidal active ingredients. Thepresent invention furthermore relates to DNA sequences encoding apolypeptide with dihydroorotase (EC 3.5.2.3) activity. Also, theinvention relates to the use of a nucleic acid encoding a protein withdihydroorotase activity of vegetable origin for the generation of a testsystem for identifying herbicidally active dihydroorotase inhibitors,and to inhibitors of plant dihydroorotase identified using these methodsor this assay system. In addition, the present invention relates to aDNA sequence encoding a polypeptide with dihydroorotase dehydrogenaseactivity and to its use as auxiliary enzyme in a molecular assay system.Furthermore, the invention relates to the use of the nucleic acidencoding plant dihydroorotase for the generation of plants with anincreased resistance to dihydroorotase inhibitors. In addition, theinvention relates to a method of eliminating undesired vegetation, whichcomprises treating the plants to be eliminated with a compound whichspecifically binds to, and inhibits the function of, dihydroorotaseencoded by a DNA sequence SEQ-ID No. 1 or by a DNA sequence hybridizingwith this DNA sequence.

Plants are capable of synthesizing their cell components from carbondioxide, water and inorganic salts.

This process is only possible by exploiting biochemical reactions forthe synthesis of organic substances. Nucleotides, being constituents ofthe nucleic acids, must be synthesized de novo by the plants.

Not only the enzyme reactions of the de novo purine biosynthesis, butalso the enzyme reactions of the de novo pyrimidine biosynthesis, areimportant for regulating the nucleotide metabolism. One of these enzymesis dihydroorotase. The enzyme catalyzes the elimination of water fromcarbamoyl aspartate and the cyclization to give dihydroorotate. Thesubsequent enzyme dihydroorotate dehydrogenase converts dihydroorotateinto orotate via a redox reaction, see FIG. 1.

Genes which encode dihydroorotases were isolated from a variety oforganisms. Complete cDNA sequences are known from bacteria (GenBank Acc.No. M97254, Pseudomonas putida, X84262 Lactobacillus leichmannii,AE000207 Escherichia coli, M97253 Pseudomonas putida, P74438Synechocystis). In eukaryotes, dihydroorotase is a component of amultifunctional enzyme complex which is localized on an coding sequence(for example X03881 Drosophila melanogaster). In yeast, too,dihydroorotase is present in a multi-enzyme complex (Souciet et al.,Mol. Gen. Genet. 207 (2-3), 314-319 (1987)). In plants, dihydroorotaseis not a component of a polyfunctional polypeptide, but, similarly towhat is the case in E. coli, exists as a separate enzyme. A plantdihydroorotase has hitherto only been isolated from Arabidopsis thaliana(Genbank Acc. No. AF000146; Zhou et al., Plant Physiol. 114 (1997),1569).

The demonstration that an enzyme is suitable as herbicide target can beshown, for example, by reducing the enzyme activity by means of theantisense technology in transgenic plants. If this results in reducedgrowth, it can be concluded that the enzyme, whose activity is reduced,is suitable as site of action for herbicidal active ingredients. Thiswas shown by way of example for acetolactate synthase in transgenicpotato plants (Höfgen et al., Plant Physiology 107 (1995), 469-477).

It is an object of the present invention to prove that dihydroorotase inplants is a suitable herbicidal [sic] target, to isolate a completeplant cDNA encoding the enzyme dihydroorotase and its functionalexpression in bacterial or eukaryotic cells, and to generate anefficient and simple test system for carrying out inhibitor-enzymebinding studies.

We have found that this object is achieved by isolating a gene encodingthe plant enzyme dihydroorotase, generating dihydroorotase antisenseconstructs, and functionally expressing dihydroorotase in bacterial oreukaryotic cells.

The present invention firstly relates to a DNA sequence SEQ-ID NO:1comprising the coding region of a plant dihydroorotase from Solanumtuberosum (potato), see Examples 1 and 2.

The present invention furthermore relates to DNA sequences which arederived from this SEQ-ID NO:1 or hybridize herewith and which encode aprotein which has the biological activity of a dihydroorotase.

Plants of the ROSa lines, which carry a dihydroorotase antisenseconstruct, have been characterized in greater detail. The plants exhibitdifferent degrees of growth retardation. The plant line ROSa-40 isaffected to such an extent that no tubers are formed. Plants of thisline are not viable in the greenhouse and must be maintained in vitro. Acorrelation between growth retardation and reduction in thedihydroorotase protein quantity can be found. This clear connectionidentifies dihydroorotase unambiguously as novel target protein forherbicidal active ingredients, see Examples 3-7.

To allow effective inhibitors of plant dihydroorotase to be found,suitable test systems must be provided with which inhibitor-enzymebinding studies can be carried out. To this end, for example, thecomplete cDNA sequence of Solanum tuberosum dihydroorotase is clonedinto an expression vector (pQE, Qiagen) and overexpressed in E. coli,see Example 8. Alternatively, however, the expression cassettecomprising a DNA sequence SEQ-ID No. 1 can be expressed, for example, inother bacteria, in yeasts, fungi, algae, plant cells, insect cells ormammalian cells.

The dihydroorotase protein expressed with the aid of the expressioncassette according to the invention is particularly suitable for findingdihydroorotase-specific inhibitors.

To this end, the dihydroorotase can be employed, for example, in anenzyme test in which the dihydroorotase activity in the presence andabsence of the active ingredient to be tested is determined. Bycomparing the two activity determinations, a qualitative andquantitative statement can be made on the inhibitory behavior of theactive ingredient to be tested.

The enzymatic detection developed hitherto for measuring thedihydroorotase activity by the method of Mazus and Buchowicz (ActaBiochimica Polonica (1968), 15 (4), 317-325) is based on detecting theorotate formed in a dihydroorotate-dehydrogenase-coupled reactionmixture at 280 nm. This assay is not suitable for mass screening. Themethod was therefore designed in such a way that NADH formed can bedetected at 340 nm. To do this, a high activity of the auxiliary enzyme,the dihydroorotate dehydrogenase, is required. A commercially availablepreparation from Zymobacterium oroticum (Sigma) proved to be too impurefor the NADH formation to be monitored. In order to be able to carry outmass screening, the specific dihydroorotate dehydrogenase activity mustbe at least ten times higher than that in the commercial preparation.Such an activity was obtained by isolating a plant dihydroorotatedehydrogenase and expressing it in yeast (Saccharomyces cerevisiae).This is why a test system was developed which was based on couplingplant dihydroorotase and plant dihydroorotate dehydrogenase. To thisend, for example the gene encoding an Arabidopsis thalianadihydroorotate/dehydrogenase was isolated (see Genbank Acc. No. x62909,Minet et al., Plant J. (1992), 2 (3), 417-422; Examples 9-11.

The test system according to the invention allows a large number ofchemical compounds to be tested simply and rapidly for herbicidalproperties. The method allows reproducibly to select in a directedfashion, from a multitude of substances, those with high potency inorder to use these substances for subsequently carrying out otherin-depth tests with which the skilled worker is familiar.

The invention furthermore relates to a method of identifyingherbicidally active substances which inhibit the dihydroorotase activityin plants, consisting of the following steps

a) the generation of transgenic plants, plant tissues or plant cellswhich comprise an additional DNA sequence encoding an enzyme withdihydroorotase activity and which are capable of overexpressing anenzymatically active dihydroorotase;

b) applying a substance to transgenic plants, plant cells, plant tissuesor plant parts and to untransformed plants, plant cells, plant tissuesor plant parts;

c) determining the growth or the viability of the transgenic and theuntransformed plants, plant cells, plant tissues or plant parts afterapplication of the chemical substance; and

d) comparing the growth or the viability of the transgenic and theuntransformed plants, plant cells, plant tissues or plant parts afterapplication of the chemical substance;

where suppression of the growth or the viability of the untransformedplants, plant cells, plant tissues or plant parts without greatlysuppressing the growth or the viability of the transgenic plants, plantcells, plant tissues or plant parts confirms that the substance of b)shows herbicidal activity and inhibits the dihydroorotase enzymeactivity in plants.

The invention furthermore relates to a method of eliminating undesiredvegetation, which comprises treating the plants to be eliminated with acompound which specifically binds to, and inhibits the function of,dihydroorotase encoded by a DNA sequence SEQ-ID No. 1 or a DNA sequencehybridizing with this DNA sequence.

The present invention furthermore relates to herbicidally activecompounds which can be identified with the above-described test system.

Herbicidally active dihydroorotase inhibitors can be employed asdefoliants, desiccants, haulm killers and, in particular, as weedkillers. Weeds are to be understood as meaning, in the broadest sense,all plants which grow in locations where they are undesired. Whether theactive ingredients found with the aid of the test system according tothe invention act as total or selective herbicides depends, inter alia,on the quantity applied.

For example, herbicidally active dihydroorotase inhibitors can be usedagainst the following weeds:

Dicotyledonous Weeds of the Genera:

Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga,Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium,Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus,Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica,Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium,Ranunculus, Taraxacum.

Monocotyledonous Weeds of the Genera:

Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca,Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum,Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis,Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis,Alopecurus, Apera.

The present invention also relates to expression cassettes whosesequences encode a Solanum tuberosum dihydroorotase or its functionalequivalent. The nucleic acid sequence can be, for example, a DNA or acDNA sequence.

In addition, the expression cassettes according to the inventioncomprise regulatory nucleic acid sequences which govern the expressionof the coding sequence in the host cell. In accordance with a preferredembodiment, an expression cassette according to the invention comprisesupstream, i.e. at the 5′-end of the coding sequence, a promoter anddownstream, i.e. at the 3′-end, a polyadenylation signal and, ifappropriate, other regulatory elements which are operably linked withthe coding sequence, for the dihydroorotase gene, which is located inbetween. Operable linkage is to be understood as meaning the sequentialarrangement of promoter, coding sequence, terminator and, ifappropriate, other regulatory elements in such a way that each of theregulatory elements can fulfill its intended function when the codingsequence is expressed.

An expression cassette according to the invention is generated by fusinga suitable promoter with a suitable dihydroorotase DNA sequence and apolyadenylation signal using customary recombination and cloningtechniques as they are described, for example, in T. Maniatis, E. F.Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and in T. J.Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and inAusubel, F. M. et al., Current Protocols in Molecular Biology, GreenePublishing Assoc. and Wiley-Interscience (1987).

The sequence homology between Solanum tuberosum dihydroorotase andArabidopsis thaliana dihydroorotase is 78% identity at protein level.The homology was obtained using the program BLASTP (Altschul et al.,Nucleic Acids Res. (1997) 25, 3389-3402), see Example 2.

The present invention also relates to functionally equivalent DNAsequences which encode a dihydroorotase gene and which, based on thetotal length of the gene, show 40 to 100% sequence homology with the DNAsequence SEQ-ID NO: 1.

Preferred subject matter of the invention are functionally equivalentDNA sequences which encode a dihydroorotase gene and which, based on thetotal length of the gene, show 60 to 100% sequence homology with the DNAsequence SEQ-ID NO: 1.

Particularly preferred subject matter of the invention are functionallyequivalent DNA sequences which encode a dihydroorotase gene and which,based on the total length of the gene, show 80 to 100% sequence homologywith the DNA sequence SEQ-ID NO: 1.

Functionally equivalent sequences which encode a dihydroorotase geneare, in accordance with the invention, those sequences which still havethe desired functions, despite a differing nucleotide sequence.Functional equivalents thus encompass naturally occurring variants ofthe sequences described herein, and also artificial, for examplechemically synthesized, artificial [sic] nucleotide sequences adapted tosuit the codon usage of a plant.

A functional equivalent is also to be understood as meaning, inparticular, natural or artificial mutations of an originally isolated,dihydroorotase-coding sequence which continues to show the desiredfunction. Mutations encompass substitutions, additions, deletions,exchanges or insertions of one or more nucleotide residues. Thus, forexample, the present invention also encompasses those nucleotidesequences which are obtained by modifying this nucleotide sequence. Theaim of such a modification can be, for example, to further delimit thecoding sequence contained therein, or else, for example, insert morerestriction enzyme cleavage sites.

Functional equivalents are also those variants whose function is weakeror stronger in comparison with the original gene or gene fragment.

In addition, the expression cassette according to the invention can alsobe employed for the transformation of bacteria, cyanobacteria, yeasts,filamentous fungi and algae with the purpose of producing sufficientamounts of the enzyme dihydroorotase.

The present invention furthermore relates to a Solanum tuberosum proteinwhich comprises the amino acid sequence SEQ-ID NO:2 or derivatives orparts of this protein with dihydroorotase activity. In comparison withthe Arabidopsis thaliana dihydroorotase, the homology at amino acidlevel is 78% identity.

The present invention also relates to plant proteins with dihydroorotaseactivity with an amino acid sequence homology to the Solanum tuberosumdihydroorotase of 20-100% identity.

Preferred plant proteins with dihydroorotase activity are those with anamino acid sequence homology to the Solanum tuberosum dihydroorotase of50-100% identity.

Particularly preferred plant proteins with dihydroorotase activity arethose with an amino acid sequence homology to the Solanum tuberosumdihydroorotase of 80-100% identity.

It is another object of the present invention to overexpress thedihydroorotase gene in plants in order to generate plants which toleratedihydroorotase inhibitors.

Overexpressing the dihydroorotase-encoding gene sequence SEQ-ID NO: 1 ina plant results in an increased resistance to dihydroorotase inhibitors.The present invention also relates to the transgenic plants generatedthus.

The expression efficacy of the transgenically expressed dihydroorotasegene can be determined, for example, in vitro by shoot meristemmultiplication, or by a germination test. Also, an altered expressiontype and expression level of the dihydroorotase gene and their effect onthe resistance to dihydroorotase inhibitors may be tested on test plantsin greenhouse experiments.

The present invention furthermore relates to transgenic plantstransformed with an expression cassette according to the inventioncomprising the DNA SEQ-ID No. 1, which plants have been made tolerant todihydroorotase inhibitors by additional expression of the DNA sequenceSEQ-ID No. 1, and to transgenic cells, tissues, parts and propagationmaterial of such plants. Especially preferred are transgenic crop plantssuch as, for example, barley, wheat, rye, maize, soybeans, rice, cotton,sugar beet, canola, sunflowers, flax, hemp, potatoes, tobacco, tomatoes,oilseed rape, alfalfa, lettuce, and the various tree, nut and grapevinespecies, and also legumes.

The invention furthermore relates to plants, which, after expression ofthe DNA SEQ ID NO:1 in the plant, show an increased UMP content.

Increasing the uridine-5′-phosphate (UMP) content means, for thepurposes of the present invention, the artificially acquired capabilityof an increased UMP biosynthesis performance by functionallyoverexpressing the dihydroorotase gene in the plant compared to thenon-genetically-engineered plant for at least one plant generation.

Especially preferred sequences are those which ensure targeting into theapoplast, into plastids, into the vacuole, into the mitochondrion orinto the endoplasmatic reticulum (ER) or which, due to a lack ofsuitable operative sequences, ensure that the product remains in thecompartment of formation, the cytosol (Kermode, Crit. Rev. Plant Sci.15, 4 (1996), 285-423).

For example, the plant expression cassette can be incorporated into thetobacco transformation vector pBinAR (see Example 3).

A suitable promoter of the expression cassette according to theinvention is, in principle, any promoter which is capable of governingthe expression of foreign genes in plants. In particular, a plantpromoter or a promoter derived from a plant virus is preferably used.Especially preferred is the cauliflower mosaic virus CaMV 35S promotor(Franck et al., Cell 21 (1980), 285-294). This promoter contains variousrecognition sequences for transcriptional effectors which in theirtotality lead to permanent and constitutive expression of the introducedgene (Benfey et al., EMBO J. 8 (1989), 2195-2202).

The expression cassette according to the invention may also comprise achemically inducible promoter which allows expression of the exogenousdihydroorotase gene in the plant to be governed at a particular point intime. Such promoters, for example the PRP1 promotor (Ward et al., Plant.Mol. Biol. (1993) 22, 361-366), a salicylic-acid-inducible promoter (WO95/1919443), a benzenesulfonamide-inducible promoter (EP 388186), atetracyclin-inducible promoter (Gatz et al., Plant J. (1992) 2,397-404), an abscisic-acid-inducible promoter (EP0335528) or an ethanol-or cyclohexanone-inducible promoter (WO 93/21334) are described in theliterature and can be used, inter alia.

Furthermore, especially preferred promoters are those which ensureexpression in tissues or parts of the plant in which the biosynthesis ofpurins or their precursors takes place. Promoters which ensureleaf-specific expression may be mentioned in particular. Promoters whichmay be mentioned are the potato cytosolic FBPase or the potato ST-LSIpromoter (Stockhaus et al., EMBO J., (1989) 8, 2445-251 [sic]).

A foreign protein can be expressed stably in the seeds of transgenictobacco plants to an extent of 0.67% of the total soluble seed proteinwith the aid of a seed-specific promoter (Fiedler and Conrad,Bio/Technology (1995) 10, 1090-1094). The expression cassette accordingto the invention can therefore comprise, for example, a seed-specificpromoter (preferably the phaseolin promotor, the USP or LEB4 promotor),the LEB4 signal peptide, the gene to be expressed, and an ER retentionsignal.

The inserted nucleotide sequence encoding a dihydroorotase can begenerated synthetically or obtained naturally or comprise a mixture ofsynthetic and natural DNA components. In general, synthetic nucleotidesequences are generated which have codons which are preferred by plants.These codons which are preferred by plants can be determined by codonswith the highest protein frequency which are expressed in most of theplant species of interest. When preparing an expression cassette, it ispossible to manipulate various DNA fragments so as to obtain anucleotide sequence which expediently reads in the correct direction andwhich is equipped with a correct reading frame. To link the DNAfragments to each other, adapters or linkers may be attached to thefragments.

Other suitable DNA sequences are artificial DNA sequences as long asthey mediate, as described above by way of example, the desired propertyof increasing the UMP content in the plant by overexpressing thedihydroorotase gene in crop plants. Such artificial DNA sequences can bedetermined, for example, by backtranslating proteins which have beenconstructed by means of molecular modeling and which exhibitdihydroorotase activity, or by in vitro selection. Especially suitableare encoding DNA sequences which have been obtained by backtranslating apolypeptide sequence in accordance with the host-plant-specific codonusage. The specific codon usage can be determined readily by a skilledworker familiar with plant-genetic-engineering methods by means ofcomputer evaluations of other, known genes of the plant to betransformed.

Further suitable equivalent nucleic acid sequences according toinvention which may be mentioned are sequences which encode fusedproteins, component of the fused protein being a plant dihydroorotasepolypeptide or a functionally equivalent portion thereof. The secondportion of the fused protein can be, for example, a furtherenzymatically active polypeptide or an antigenic polypeptide sequencewith the aid of which detection for dihydroorotase expression ispossible (for example myc-tag or his-tag). However, it is preferably aregulatory protein sequence such as, for example, a signal or transitpeptide, which leads the dihydroorotase protein to the desired site ofaction.

Expediently, the promoter regions according to the invention and theterminator regions should be provided, in the direction oftranscription, with a linker or polylinker comprising one or morerestriction sites for insertion of this sequence. As a rule, the linkerhas 1 to 10, in most cases 1 to 8, preferably 2 to 6, restriction sites.In general, the linker within the regulatory regions has a size lessthan 100 bp, frequently less than 60 bp, but at least 5 bp. The promoteraccording to the invention can be native, or homologous, or elseforeign, or heterologous, to the host plant. The expression cassetteaccording to the invention comprises, in the 5′-3′-direction oftranscription, the promoter according to the invention, any sequence anda region for transcriptional termination. Various termination regionsmay be exchanged for each other as desired.

Manipulations which provide suitable restriction cleavage sites or whicheliminate the excess DNA or excess restriction cleavage sites may alsobe employed. In vitro mutagenesis, prime repair, restriction or ligationmay be used in cases where insertions, deletions or substitutions suchas, for example, transitions and transversions, are suitable.Complementary ends of the fragments may be provided for ligation in thecase of suitable manipulations such as, for example, restriction,chewing back or filling in overhangs for blunt ends.

Preferred polyadenylation signals are plant polyadenylation signals,preferably those which correspond essentially to Agrobacteriumtumefaciens T-DNA polyadenylation signals, in particular those of gene 3of the T-DNA (octopine synthase) of the Ti-plasmid pTiACH5 (Gielen etal., EMBO J. 3 (1984) 835 ff), or functional equivalents.

For transforming a host plant with a dihydroorotase-encoding DNA, anexpression cassette according to the invention is incorporated, asinsertion, into a recombinant vector whose vector DNA comprisesadditional functional regulatory signals, for example sequences forreplication or integration. Suitable vectors are described, inter alia,in “Methods in Plant Molecular Biology and Biotechnology” (CRC Press),Chapter 6/7, pp. 71-119.

The transfer of foreign genes into the genome of a plant is termedtransformation. It exploits the above-described methods for transformingand regenerating plants from plant tissues or plant cells for transientor stable transformation. Suitable methods are protoplast transformationby polyethylene glycol-induced DNA uptake, the biolistic method usingthe gene gun, electroporation, incubation of dry embryos inDNA-containing solution, microinjection and agrobacterium-mediated genetransfer. The abovementioned methods are described in, for example, B.Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol.1, Engineering and Utilization, edited by S. D. Kung and R. Wu, AcademicPress (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. PlantMolec. Biol. 42 (1991) 205-225). The construct to be expressed ispreferably cloned into a vector which is suitable for the transformationof Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl.Acids Res. 12 (1984) 8711).

Agrobacteria transformed with an expression cassette according to theinvention can equally be used in a known manner for transforming plants,in particular crop plants such as cereals, maize, soybeans, rice,cotton, sugar beet, canola, sunflowers, flax, hemp, potatoes, tobacco,tomatoes, oilseed rape, alfalfa, lettuce and the various tree, nut andgrapevine species, and legumes, for example by bathing wounded leaves orleaf sections in an agrobacterial suspension and subsequently growingthem in suitable media.

The biosynthesis site of pyrimidines is, generally, the leaf tissue, sothat leaf-specific expression of the dihydroorotase gene is useful.However, it is obvious that the pyrimidine biosynthesis need not belimited to the leaf tissue, but may also take place in all otherremaining parts of the plant in a tissue-specific fashion, for examplein fatty seeds.

Moreover, constitutive expression of the exogenous dihydroorotase geneis advantageous. On the other hand, inducible expression may also bedesirable.

Using the above-cited recombination and cloning techniques, theexpression cassettes according to the invention can be cloned intosuitable vectors which allow them to be multiplied, for example in E.coli. Suitable cloning vectors are, inter alia, pBR332, pUC series, M13mp series and pACYC184. Especially suitable are binary vectors, whichare capable of replication both in E. coli and in agrobacteria.

The present invention furthermore relates to the use of an expressioncassette according to the invention for the transformation of plants,plant cells, plant tissues or parts of plants. The preferred aim of theinvention is to increase the dihydroorotase content in the plant.

Depending on the choice of the promoter, expression may take placespecifically in the leaves, in the seeds or other parts of the plant.Such transgenic plants, their propagation material and their plantcells, tissue or parts are a further subject of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary methods and arrangements conducted and configured according tothe advantageous solutions presented herein are depicted in theaccompanying drawings wherein:

FIG. 1 is a flow diagram illustrating the enzymatic conversion ofdihydroorotate into orotate via a redox reaction by dihydroorotatedehydrogenase;

FIG. 2 is an illustration of dihydroorotase protein quantity in leaf andtuber of selected transformants of line ROSa; and,

FIG. 3 is an illustration of Dihydroorotase mRNA content in fully grownleaves of selected transformants of line ROSa.

The invention is illustrated by the examples which follow, but notlimited thereto:

EXAMPLES

Genetic engineering methods on which the use examples are based:

General Cloning Methods

Cloning methods such as, for example, restriction cleavage, agarose gelelectrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linking DNA fragments,transformation of Escherichia coli cells, growing bacteria, and thesequence analysis of recombinant DNA, were carried out as described bySambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN0-87969-309-6).

Sequence Analysis of Recombinant DNA

Recombinant DNA molecules were sequenced using an ABI laser fluorescenceDNA sequencer following the method of Sanger (Sanger et al. (1977),Proc. Natl. Acad. Sci. USA74, 5463-5467). Fragments resulting from apolymerase chain reaction were sequenced and checked in order to avoidpolymerase errors in constructs to be expressed.

Example 1

Isolation of a cDNA Encoding a Functional Plant Dihydroorotase

A clone encoding dihydroorotase was obtained from potatoes by functionalcomplementation of an E. coli mutant. The mutant used was the mutantCGSC5152 (CS101-2U5) of the E. coli Genetic Stock Center, which carriesa mutation in the pyrC gene locus encoding a dihydroorotase.Complementation was effected by electrotransformation of competent cellsof strain CGSC5152 with a cDNA library in the vector plasmid pBS SK—.The underlying lambda ZAPII library (Stratagene) was cloned in anundirected fashion with EcoRI/NotI linkers following standardprocedures. The RNA template for the cDNA was isolated from sink leaves(small 1-cm-leaflets harvested from 10-week-old potato plants, grown inthe greenhouse).

The transformed E. coli cells were plated on M9 minimal medium (Sambrooket al., 1989) complemented with methionine (20 mg/l), ampicillin (100mg/l) and IPTG (2.5 mM). In total, 4 micrograms of the library weretransformed in 8 batches, giving rise to 36 clones which, followingexamination by means of restriction cleavage, proved to be identical.

Example 2

Sequence Analysis of the cDNA Clones Encoding a Protein withDihydroorotase Activity

The resulting 36 cDNA clones encode a polypeptide with homology todihydroorotases from other organisms. The homology was obtained usingthe program BLASTP (Altschul et al., Nucleic Acids Res. (1997) 25,3389-3402). Accordingly, the protein has 78% identity with Arabidopsisthaliana dihydroorotase, 58% identity with Synechocystis dihydroorotase,55% identity with E. coli and Pseudomonas putida dihydroorotase. Thelongest clone was termed pyrCSt5. The plasmid was given the namepBSSK-pyrCSt5. The cDNA (see SEQ-ID No. 1) has an open reading frame of1046 base pairs with a stop sodon in position 1047-1049. The amino acidsequence starts with the third base in the reading frame and can betranslated into a polypeptide 348 amino acids in length (see SEQ-ID No.2). This corresponds to the length of prokaryotic dihydroorotase-codingsequences.

Owing to the reading frame of the present cDNA sequence, it cannot bededuced with certainty whether it might possibly be a form localized inthe plastids or a cytosolic form.

Example 3

Generation of Plant Expression Cassettes

A35S CaMV promoter was inserted into plasmid pBin19 (Bevan et al., Nucl.Acids Res. 12 (1980), 8711) in the form of an EcoRI-KpnI fragment(corresponding to nucleotides 6909-7437 of the cauliflower mosaic virus(Franck et al., Cell 21 (1980), 285). The polyadenylation signal of gene3 of the T-DNA from Ti-plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984),835), nucleotides 11749-11939 was isolated as a PvuII-HindIII fragmentand, after addition of SphI linkers, cloned into the PvuII cleavage sitebetween the SphI-HindIII cleavage site of the vector. This gave rise toplasmid pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990),221-230). Cloning of a construct of pyrCSt5 in antisense orientation inpBinAR was done by an Asp718 cleavage site (internal cleavage site of964 bp) and a BamHI cleavage site (from the polylinker).

Example 4

Generation of Transgenic Potato Plants

Potato plants (cv. Solara) were transformed with the aid ofAgrobacterium tumefaciens using the corresponding constructpBinAR-anti-pyrCSt5. The plasmid was transformed into Agrobacteriumtumefaciens C58C1:pGV2260 (Deblaere et al., Nucl. Acids. Res. 13 (1984),4777-4788). To transform potatoes by the method of Rocha-Sosa et al.(EMBO J., 8 (1988), 23-29), a 1:50 dilution of an overnight culture of apositively transformed agrobacterial colony in Murashige-Skoog medium(Physiol. Plant., 15 (1962), 473) was used. Leaf disks of sterile plants(in each case approx. 1 cm²) were incubated for 5-10 minutes in a 1:50agrobacterial solution in a petri dish. This was followed by incubationin the dark for 2 days at 205 C on MS medium. Cultivation wassubsequently continued in a 16 hour light/8 hour dark photoperiod. Forshoot induction, explants were transferred weekly to MS mediumsupplemented with 500 mg/l claforan (cefotaxime-sodium), 50 mg/lkanamycin and plant hormones (Rocha-Sosa et al., EMBO J., 8, 23-29,1989) and 1.6 g/l glucose. Growing shoots were transferred to MS mediumsupplemented with 2% sucrose, 250 mg/l claforan and 0.8% Bacto-agar.

Regenerated shoots are obtained on 2 MS medium supplemented withkanamycin and claforan, transferred into the soil after they have struckroots and, after culture for two weeks in a controlled-environmentcabinet in a 16-hour-light/8-hour-dark photoperiod at an atmospherichumidity of 50%, examined for expression of the foreign gene, alteredmetabolite contents and phenotypic growth characteristics. Alterednucleotide contents may be determined, for example, by the method ofStitt et al. (FEBS Letters, 145 (1982), 217-222).

Example 5

Analysis of Total RNA from Plant Tissues

Total RNA from plant tissues was isolated as described by Logemann etal., Anal. Biochem. 163 (1987), 21. For the analysis, in each case 20micrograms of RNA were separated in a formaldehyde-containing 1.5%strength agarose gel and transferred to Duralon UV membranes(Stratagene).

To detect specific transcripts, digoxygenine-labeled probes wereprepared by means of PCR following the manufacturer's instructions andused for hybridization (DIG EasyHyb, Boehringer). Then, the membraneswere washed for 3×20 minutes in wash buffer (2×SSC, 0.1% SDS) at 605 C.Detection was carried out by luminescence and exposure to Hyperfilm ECL(Amersham) using the Boehringer DIG detection system with CDP-Star assubstrate.

Resulting individual transgenic plants of lines ROSa-34, -31, -10, -19,-9 and -3 are shown in FIG. 3 as test plants at RNA level. A band isrecognizable at 1.6 kb in accordance with the expected dihydroorotasetranscript size and, in the case of plants ROSa-3, -9, -31, -34, the 1.1kb antisense transcript. A marked reduction in RNA quantity can befound, in particular, in the case of plant ROSa-9.

Example 6

Detection of the Potato Dihydroorotase Protein in Tuber and Leaf Tissues

To generate a polyclonal serum against the dihydroorotase polypeptide, apeptide sequence from the potato dihydroorotase amino acid sequence waschosen. The peptide LGTDSAPHDRRRKEC (SEQ ID NO: 5) was synthesized by acommercial company (Eurogentec, Seraing, Belgium) and coupled to KLH(keyhole limpet protein) via the C-terminal cysteine. The conjugate wasemployed, again, by the commercial company (Eurogentec) for immunizingrabbits and antisera against the peptide were obtained. In Western blotexperiments, the antiserum specifically recognizes the potatopolypeptide. To this end, protein was subjected to an SDS polyacrylamidegel electrophoresis under denaturing conditions, transferred tonitrocellulose membranes and detected by means of immunodetectionfollowing the manufacturer's instructions (ECL-System, Amersham).Transgenic plants of the ROSa lines were characterized with the aid ofthe antiserum. Lines-3, -9 and -40 show different degrees of proteinreduction in the leaf, see FIG. 2. Plant-40 does not form tubers.Plants-3 and -9 also show a correspondingly greatly reduceddihydroorotase protein quantity in tubers.

Example 7

Phenotypic Analysis of Transgenic Plants

Plants of lines ROSa, which carry a dihydroorotase antisense constructwere characterized in greater detail. The plants show differing degreesof growth retardation. Plant line ROSa-40 is affected to such an extentthat no tubers are formed. Plants of this line are not viable in thegreenhouse and must be maintained in vitro. A correlation can be foundbetween growth retardation and reduction in dihydroorotase proteinquantity. This clear connection identifies potato dihydroorotaseunambiguously as novel target protein for herbicidal active ingredients.

Example 8

Generation of Overexpression Vectors in E. coli

The following oligonucleotide sequences were derived from the sequencedetermined, and provided with a BamHI restriction cleavage site and withtwo base overhangs.

(SEQ ID NO: 6) 1. 5′-primer aaggatccGCAAAAATGGAGCTCTCA (SEQ ID NO: 7) 2.3′-primer aaggatccTCAGAGAGGAGCCGGCAAC

The PCR reaction mixtures contained 8 ng/ml pBSSK-pyrCSt5 DNA, 0.5 mM ofthe corresponding oligonucleotides, 200 mM nucleotides (Pharmacia), 50mM KCl, 10 mM Tris-HCl (pH 8.3 at 25° C.), 1.5 mM MgCl₂ and 0.02 U/mlTaq polymerase (Perkin Elmer). The amplification conditions were set asfollows:

Denaturation temperature: 92° C., 1 min Annealing temperature: 52° C., 1min Elongation temperature: 72° C., 2.5 min Number of cycles: 30

The PCR fragments were cloned into the overexpression vector pQE9 viaBamHI and employed for protein production by means of IPTG inductionfollowing standard methods (see Handbuch: The QiaExpressionist, Qiagen,Hilden).

Example 9

Test System for Measuring the Dihydroorotase Activity

The enzymatic detection developed to date for measuring thedihydroorotase activity by the method of Mazus and Buchowicz, (ActaBiochimica Polonica (1968), 15(4), 317-325) is based on detecting theorotate formed at 280 nm in a dihydroorotate-dehydrogenase-coupledreaction mixture. Prerequisite for doing so is a high activity of theauxiliary enzyme, viz. dihydroorotate dehydrogenase. A commerciallyavailable preparation from Zymobacterium oroticum (Sigma) proved to betoo contaminated.

In order to be able to carry out a mass screening, the specificdihydroorotate dehydrogenase activity must be at least ten times higherthan is the case in the commercial preparation. Such an activity wasobtained by preparing a dihydroorotate dehydrogenase activity fromNeurospora crassa (R. W. Miller, Methods in Enzymology LI, 1978, 63-69)after cloning a plant dihydroorotate dehydrogenase and its expression inyeast (Saccharomyces cerevisiae). A further improvement of the testsystem was achieved by carrying out the measurement at 340 nm n.

First, an Arabidopsis thaliana dihydroorotate dehydrogenase was isolated(see Genbank Acc. No. X62909, Minet et al., Plant J. (1992), 2 (3),417-422).

The following oligonucleotide sequences were derived from the databaseentry of the dihydroorotate dehydrogenase sequence:

(SEQ ID NO: 8) 1. 5′-primer aaggatccatggccggaagggctg (SEQ ID NO: 9) 2.3′-primer aaggatccttagtggtggtggtggtggtgtttgtggg atggggc

The PCR reaction mixtures contained 10 ng of plasmid DNA from anArabidopsis thaliana cDNA in vector pFL61 (ATCC 77600), 0.5 microM [sic]of the corresponding oligonucleotides, 200 mM nucleotides (Pharmacia),50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25° C.), 1.5 mM MgCl₂ and 0.02 U/mlTaq polymerase (Perkin Elmer). The amplification conditions were set asfollows:

Denaturation temperature: 92° C., 0.5 min Annealing temperature: 52° C.,0.5 min Elongation temperature: 72° C., 1.5 min Number of cycles: 35

The resulting PCR fragment was first cloned into the yeast expressionvector pYES2 (Invitrogen) via the BamHI cleavage sites. The constructgenerated was named pYES2-pyrDAt.

Example 10

Cloning of a Plant Dihydroorotate Dehydrogenase from Tobacco

Furthermore, the PCR fragment described in Example 9 was applied for aheterologous screening in a tobacco phage cDNA library. The cDNAemployed for generating the tobacco phage cDNA library was obtained fromRNA from tobacco cell suspension cultures. The cDNA library wasgenerated following the manufacturer's instructions (Stratagene).3.0×10⁵ lambda phages of the Nicotiana tabacum cDNA library were platedon agar plates with E. coli XLI-Blue as bacterial strain.

The phage DNA was transferred to nylon filters (Duralon UV Stratagene)by means of standard methods (Sambrook et al. (1989); Cold Spring HarborLaboratory Press: ISBN 0=87969-309-6) and fixed on the filters. Thehybridization probe used was the above-described PCR fragment, which wasDIG-labeled with the aid of the labeling and detection system(Boehringer, Mannheim) following the manufacturer's instructions.Hybridization of the membrane was carried out for 16 hours at 425 C inDIG EasyHyb (Boehringer). The filters were subsequently washed for 3×20minutes in 2×SSC, 0.1% SDS at 605 C. Positively hybridizing phages wereon Hyperfilm ECL (Amersham) by luminescence with the Boehringer DIGdetection system using CDP-Star as substrate, and purified and isolatedby standard techniques.

Ten identical clones resulted, of which clone pyrDT10 was sequencedcompletely (SEQ-ID No. 3). An EcoRI digest of the clone shows an EcoRIfragment 1962 base pairs in size with an open reading frame of 458 aminoacids, a start codon in position 305-307 and a stop codon in position1679-1681. The deduced amino acid sequence (SEQ-ID No. 4) of the tobaccodihydroorotate dehydrogenase exhibits 72% identity with the Arabidopsisamino acid sequence, 51% identity with the rat amino acid sequence, 43%identity with the yeast amino acid sequence, 37% identity with the E.coli amino acid sequence. The identity was obtained using the programBLASTP (Altschul et al., Nucleic Acids Res. (1997) 25, 3389-3402).

The following oligonucleotide sequences were derived from the sequencedetermined, and provided with a KpnI restriction cleavage site and twobase overhangs.

(SEQ ID NO: 10) 1. 5′-primer ggggtaccatgagacaaagggttggatt (SEQ ID NO:11) 2. 3′-primer ggggtaccttagtggtggtggtggtggtggagaggag ccggcaacca

The PCR reaction mixtures contained 5 ng/ml pBSSK-pyrDT10 DNA, 0.5 mM ofthe corresponding oligonucleotides, 200 mM nucleotides (Pharmacia), 50mM KCl, 10 mM Tris-HCl (pH 8.3 at 25° C.), 1.5 mM MgCl₂ and 0.02 U/mlTaq polymerase (Perkin Elmer). The amplification conditions were set asfollows:

Denaturation temperature: 92° C., 1 min Annealing temperature: 52° C., 1min Elongation temperature: 72° C., 2.5 min Number of cycles: 30

The PCR fragment of the tobacco dihydroorotate dehydrogenase was clonedinto the yeast expression vector pYES2 (Invitrogen) via KpnI cleavagesites. This construct (pYES-pyrDT10) and the Arabidopsis dihydroorotatedehydrogenase construct pYES2-pyrDAt were inserted into the ural yeastmutant for complementation (Minet et al., Gene (1992), 121(2), 393-6).Resulting yeast clones were grown in liquid culture overnight incomplete medium supplemented with 1% galactose.

Example 11

Enzyme Isolation of Plant Dihydroorotase and DihydroorotateDehydrogenase, and Measurement of the Dihydroorotase Activity

The dihydroorotase E. coli expression cultures, and the yeast expressionculture containing the tobacco (or Arabidopsis) dihydroorotatedehydrogenase, were in each case disrupted separately by means ofpressure disruption methods using the French Press under maximumpressure in a 20 ml pressurized chamber, or with the aid of a glass ballmill (IMA Disintegrator). Per 1 g of cell pellet, 10 ml of buffer (0.1 MKH₂PO₄; pH 7.5; 0.4M sucrose, 0.1 mM DTT) are used. By adding a 2.5-foldamount of glass beads (d=0.5 mm), the pellet is disrupted in the glassball mill for 20 minutes at 45 C and 2500 rpm. The batch is centrifugedfor 20 minutes at 45 C and 100,000 g. The enzyme activity was determinedin a photometric assay by measurement in a photometer (Uvikon 933,Kontron) at 340 nm. The choice of the overexpression vectors alsoallowed the dihydroorotase and the dihydroorotate dehydrogenase to bepurified via the histidin anchor by standard methods in one step undernative conditions if the disruption buffer was free from DTT (cf. alsoHandbuch: The QiaExpressionist, Qiagen, Hilden). The eluates weresubjected to dialysis to change the buffer to 20 mM potassium phosphatebuffer pH 6.1; 5 mM MgCl₂; 1 mM DTT; 10 mM cysteine; 10 mM ZnCl₂, 20 mMNAD. In each case 10-100 ml of the resulting enzyme fraction was made upwith buffer to 700 ml and measured against a reference cell containing700 ml reaction buffer and 100 ml of a protein homogenate ofuntransformed E. coli culture. The reaction was started using 7 mMcarbamyl aspartate. Identical quantities of total protein were employedfor measuring the untransformed or transformed E. coli extracts.

As an alternative to plant dihydroorotate dehydrogenase activitiesexpressed in yeasts, it is possible to employ a dihydroorotatedehydrogenase activity prepared from Neurospora crassa, see R. W.Miller, Dihydroorotate dehydrogenase, (in: Methods in Enzymology 51(1978), 63-69).

Alternatively, the dihydroorotase may also be measured in a lesssensitive colorimetric assay by the method of Prescott and Jones (Anal.Biochem. (1969) 32, 408-419) without being coupled to dihydroorotatedehydrogenase. To this end, the dihydroorotase activity was measured in50 mM Tris-HCl, 1 mM dihydroorotate (pH 8.5) after incubation at 375 Cby detecting the carbamoyl aspartate formed. Prerequisite to this is theprotein preparation with high protein activity which has been describedin this example.

The potato dihydroorotase activity measured in the assay systemsdescribed can be reduced with known dihydroorotase inhibitors such as6-L-thiodihydroorotate or2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylate (Christopherson etal., Biochemical Society Transactions 23: 888-893, 1995).

1. An isolated DNA sequence encoding a polypeptide having dihydroorotaseactivity, the isolated DNA sequence comprising one of: SEQ ID NO: 1; or,a sequence having at least 95% identity with SEQ ID NO:
 1. 2. A methodof synthesizing a dihydrooratase comprising: introducing the isolatedDNA sequence of claim 1 into a prokaryotic or eukaryotic organism;linking the isolated DNA sequence to regulatory elements to ensuretranscription and translation of the isolated DNA in cells of theorganism; and, expressing translatable mRNA corresponding to theisolated DNA sequence to cause the synthesis of the dihydroorotase.